Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.     

Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis.

Infection of African green monkey kidney cells with type 5 adenovirus leads to the synthesis of two infected, cell-specific proteins with approximate molecular weights of 72,000 and 48,000, that bind specifically to single-stranded but not double-stranded DNA. The production of these two proteins was studied after infection with two DNA-negative adenovirus mutants belonging to different complementation groups (H5 ts36 and H5 ts 125). Both DNA binding proteins were detected in cells infected with either mutant at the permissive temperature (32 C) AND ALSO IN H5 ts36-infected cells at the nonpermissive temperature (39.5 C). In H5 ts125-infected cells at 39.5 C, however, less than 5% of the normal wild-type level of these DNA binding proteins was detectable. When H5 ts125-infected cells were labeled with radioactive leucine at 32 C and subsequently shifted to 39.5 C in the presence of unlabeled leucine (chase), the level of DNA binding proteins found in these infected cells was markedly reduced compared to cultures not shifted to 39.5 C. These data suggest that the DNA binding proteins themselves were temperature sensitive. This conclusion was confirmed by experiments in which the DNA binding proteins were eluted from DNA cellulose with buffers of increasing temperatures (thermal elution). The H5 ts 125 proteins were shown to elute at lower temperatures than either wild-type or H5 ts36 proteins. These results are taken to indicate that the H5 ts125 mutant codes for a DNA binding protein that is thermolabile for continued binding to single-stranded DNA.     

Adenovirus-induced alterations of the cell growth cycle: effects of mutations in early regions E2A and E2B.

Mutants ts125 (E2A) and ts36 (E2B) of adenovirus type 5 induced alterations to cell cycle progression at the nonpermissive temperature which were detectable by flow cytometry. Thus neither E2A, nor gene N in E2B, is required for these effects. Whereas the wild-type virus induced cells with aneuploid (between 4n and 8n) DNA contents, as did ts125 at the permissive temperature, ts125 induced peaks of cells with 8n, 16n, and 32n DNA contents at the nonpermissive temperature. This was probably due to the failure of regulation of E1A by E2.     

Alterations to controls of cellular DNA synthesis by adenovirus infection.

Human adenovirus type 5 and temperature-sensitive mutants ts36, ts37, and ts125 induced cellular DNA synthesis in quiescent rodent cells at both permissive and nonpermissive temperatures. Cellular DNA synthesis induced by adenovirus type 5 or by serum required protein synthesis for both initiation and continuation, whereas viral DNA synthesis was not dependent upon continued protein synthesis once it was initiated. Both cellular and viral DNA replication was induced in adenovirus type 5-infected cells in the presence of dibutyryl cyclic AMP at concentrations which inhibited induction by serum which suggested that some of the controls of DNA synthesis in serum-treated and virus-infected cells are different. After adenovirus infection of quiescent cells, there was a decrease in the number of cells with G1 DNA content and an increase in cells with G2 diploid and greater DNA contents. Thus, adenovirus type 5 induces a complete round of cellular DNA replication, but in some cells, it induces a second round without completion of a normal mitosis. These results suggest that adenovirus type 5 is able to alter cell growth cycle controls in a way which may be related to its ability to transform cells.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein.

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells.

Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.